Cartilaginous tissues perform specialized functions under normal physiological conditions. Anomalous mechanical loading of these tissues often leads to pathology. For instance, lack of mechanical stimulation of a joint leads to suppression of proteoglycan synthesis and degradation of cartilage matrix components. High and non-physiological loading of a joint contributes to the development of osteoarthritis. In order to provide effective treatments for conditions such as these, a thorough understanding of the molecular mechanisms controlling the response of cartilaginous tissues to their mechanical environment is imperative. Not withstanding, there is a dearth of knowledge about the modes of mechanical signal transduction in chondrocytes. The primary goal of this proposal is to elucidate the molecular mechanisms through which mechanical stimuli modulate the expression of cartilage extracellular matrix (ECM) components. The hypotheses to be tested are: 1) receptor-mediated cell-ECM adhesion contributes to the transduction of mechanical signals in chondrocytes; 2) mechanical signal transduction in chondrocytes requires activation of the phosphoinositol and/or cyclic AMP signaling pathways; and 3) mechanical stimulation of the expression of aggrecan is mediated through activation of specific cis-acting elements of the promoters and/or UTRs of the aggrecan gene. To test these hypotheses, experiments are designed to achieve the following Specific Aims: 1) determine the roles of integrins in modulation of aggrecan gene expression by compression; 2) determine the effect(s) of selectively inhibiting key enzymes of the phosphoinositol and cyclic AMP pathways on modulation of the aggrecan gene by mechanical loading; 3) determine whether modulation of aggrecan gene expression by mechanical loading is Ca2+-dependent; 4) determine the effects of mechanical loading on the activities of the promoter and UTRs of the human aggrecan gene; and 5) identify regions of the promoter and/or UTRs that are responsive to mechanical loading and determine their abilities to confer mechano-responsiveness to a heterologous promoter. For the loading tests, bovine articular cartilage explants will be used in Specific Aims 1 to 3, while transfected human chondrocytes in a three-dimensional matrix will be used in Specific Aims 4 and 5.